A novel organic-inorganic hybrid containing allyl benzoxazine and polyhedral oligomeric silsesquioxane (POSS) was synthesized by the thiol-ene (click) reaction. The benzoxazine (BOZ)-containing POSS (SPOSS-BOZ) copolymerized with benzoxazine/epoxy resin was used to prepare composites of SPOSS-PBZ-E nanocomposites(NPs). The polymerization behavior was monitored by FTIR and non-isothermal differential scanning calorimetry (DSC), which showed that the composites had completely cured with multiple polymerization mechanisms according to the oxazine ring-opening and epoxy resin (EP) polymerization. The thermal properties of the organic–inorganic polybenzoxazine (PBZ) nanocomposites were analyzed by DSC and thermogravimetric analysis (TGA). Furthermore, the X-ray diffraction analysis and the scanning electron microscopy (SEM) micrographs of the SPOSS-PBZ-E nanocomposites indicated that SPOSS was chemically incorporated into the hybrid nanocomposites in the size range of 80–200 nm. The flame retardancy of the benzoxazine epoxy resin composites was investigated by limiting oxygen index (LOI), UL 94 vertical burn test, and cone calorimeter tests. When the amount of SPOSS reached 10% or more, the vertical burning rating of the curing system arrived at V-1, and when the SPOSS-BOZ content reached 20 wt %, the thermal stability and flame retardancy of the material were both improved. Moreover, in the cone calorimeter testing, the addition of SPOSS-BOZ hindered the decomposition of the composites and led to a reduction in the peak heat release rate (pHRR), the average heat release rate (aHRR), and the total heat release (THR) values by about 20%, 25%, and 25%, respectively. The morphologies of the chars were also studied by SEM and energy dispersive X-ray spectroscopy (EDX), and the flame-retardant mechanism of POSS was mainly a condensed-phase flame retardant. The ceramic layer was formed by the enrichment of silicon on the char surface. When there are enough POSS nanoparticles, it can effectively protect the combustion of internal polymers.  Benzoxazine has a stable aromatic ring structure and can be cross-linked under certain conditions to form a structure such as a phenolic resin. . During Polyhedral oligomeric silsesquioxane (POSS) is a novel organic–inorganic hybrid filler with a cage structure, which is mainly composed of three-dimensional cage-like structures connected by Si–O–Si bonds and is very stable. The incorporation of POSS into benzoxazine monomers can improve the thermal, mechanical ,14,15,163-POSS) and 3,4-dihydro-3-(prop-2-ynyl)-2H-benzoxazine (P-pa) by a click reaction. However, the preparation of octa-azido functionalized POSS is too complicated. In previous studies, no researchers used thiol-based POSS and allyl benzoxazine to obtain POSS-BOZ nanocomposites nor did they study their effects on the thermal stability and flame retardancy of BOZ/E composites.It has been increasingly recognized that “good” reactions for making POSS-related materials including the criteria of efficiency, versatility, and selectivity; therefore, “click” reactions are ideal tools . Li et aTherefore, in this paper, an organic–inorganic hybrid benzoxazine was prepared by the thiol-ene (click) reaction between benzoxazine and sulfur-containing POSS. The nanocomposites were blended with benzoxazine containing allyl group (BOZ)/epoxy composites and cured. The effect of POSS on the thermal stability and flame retardancy of the material was studied.Allylamine was obtained from the Chengdu Huaxia Chemical Reagent Co. Ltd. , >95%. Paraformaldehyde was obtained from the Tianjin Fuchen Chemical Reagent Factory . Phenol was obtained from the Beijing Chemical Plant . The SPOSS (TH-1550) was obtained from Hybrid Plastics Inc. , >99%. DMPA was purchased from Aladdin Biochemical Technology Co. Ltd. , >99%. Tetrahydrofuran was obtained from Beijing Tongguang Fine Chemical Company . Epoxy resin was purchased from E-44, Beijing Tongguang Fine Chemical Company .2 atmosphere. Then, phenol  was added into the reaction system. The mixture was stirred at 80 °C for 2 h. Subsequently, the solution was cooled to room temperature, and washed using 1 mol/L NaOH solution and distilled water several times. Then, the organic layer was dried over anhydrous Na2SO4. The solution was dried under vacuum to afford a light-yellow liquid. Yield: 56 g, 80%; 1H NMR : δ 6.80–7.13 , 5.92 , 5.27–5.18 , 4.88 , 4.00 , 3.40 . FTIR (cm−1): 3077 cm−1 (stretching of =C–H), 1843–1560 cm−1 (skeleton vibration of benzene ring), 1646 cm−1 (stretching of C=C), 914 cm−1 (symmetric stretching of C-O-C), 746 cm−1 (out-of-plane bending vibrations of olefinic =C–H). HRMS (MALDI-TOF): m/z 175 .The synthesis route of BOZ is shown in Benzoxazine POSS was synthesized using the procedure presented in Composites (SPOSS-PBZ-E) containing polybenzoxazine, POSS, and epoxy resin were prepared by the procedure in 2.UV-curing: A HWUV0133X three-dimensional UV curing box  was used for the experiment. The wavelength of the ultraviolet light was 300–400 nm, the lamp power was 400W, and the radiation intensity was 100 mW/cm1H-NMR spectra were recorded on a Bruker Avance 600 NMR spectrometer  operated in the Fourier transform mode. CDCl3 was used as the solvent, and the solution was measured with tetramethyl silane (TMS) as an internal reference.Nuclear magnetic resonance (NMR) spectroscopy: −1.Fourier-transform infrared (FTIR) spectroscopy: FTIR spectra were recorded on a NICOLET 6700 IR spectrometer . The spectra were collected at 32 scans with a spectral resolution of 4 cmMass spectrometry: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed using a Bruker BIFLEX III device  equipped with a pulsed nitrogen laser . Samples were measured in positive ion modes with usually 50 spectra accumulated. For the MALDI mass spectrum, we used a-Cyano-4-hydroxycinnamic acid matrix and the salts were a mixture of NaCl and KCl. X-ray diffraction (XRD). The samples were scanned at a speed of 8 °C/min at ambient temperature using an X-ray diffractometer  at a generator voltage of 35 kV and a current of 25 mA. The data were collected from 2° to 40° intervals.Thermogravimetric analysis (TGA) was performed with a NETZSCH 209 F1 thermal analyzer  at a heating rate of 10 °C/min under nitrogen and air atmosphere, respectively. The temperature ranged from 40 to 800 °C.Differential scanning calorimetry (DSC) curves of the EP composites were measured using a Netzsch 204 F1 differential scanning calorimeter  with a pressure cell. Samples (5–10 mg) were tested at a heating rate of 10 °C/min and results from the second heating were in the range of 35–300 °C. The dynamic mechanical(DMA) properties of the cured blends were performed using a METTLER TOLEDO SDTA861 instrument  with a sample dimension of 5 × 5 × 3 mm in a controlled strain tension mode and a temperature ramp rate of 4 °C/min from −70 to 200 °C at a frequency of 1.0 Hz.The limiting oxygen index (LOI) was obtained using the standard ASTM D 2863 procedure, which involves measuring the minimum oxygen concentration required to support candle-like combustion of plastics. An oxygen index instrument  was used on barrel-shaped samples with the dimensions of 100 × 6.5 × 3 mmVertical burning tests were performed using the UL 94 standard on samples with the dimensions of 125 × 13 × 3.2 mm with the CZF-5A horizontal vertical burning tester by Jiangning Analytical Instrument Factory . In this test, the burning grade of a material was classified as V-0, V-1, V-2, or NR (no rating), depending on its behavior (dripping and burning time).Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) images were taken with a Hitachi SU8020  with a 20 kV accelerating voltage. The samples were coated with thin layers of gold to make the surface conductive. 2, according to the ISO 5660 protocol, using a Fire Testing Technology apparatus  with a truncated cone-shaped radiator. The specimen (100 × 100 × 3 mm) was measured horizontally without any grids. Typical results from the cone calorimeter were reproducible within ±10%, and the reported parameters were the average of three measurements.Cone calorimeter measurements were performed at an incident radiant flux of 50 kW/m1H NMR . Moreover, the molecular quality of P-ala was obtained in Different synthesis methods can be used to prepare benzoxazine monomers. Agag and Takeichi prepared monomers with a solventless method ; However1H NMR . The res−1 disappeared after SPOSS was modified by benzoxazine. The chemical structure of SPOSS-BOZ was confirmed by 1H NMR , 0.93 ppm (Peak a), and 1.88 ppm were caused by the seven isobutyl hydrocarbon substituents of the POSS. Next, we magnified the spectra of the allyl moiety  has been prepared using different methods ,33. Howey 1H NMR . The resl moiety B. FurtheTi, Tp, and Tf were 144 °C, 210 °C, and 240 °C, respectively, and the resin system started to react in the range of 144 to 210 °C. Combined with the relevant data [−1 and 916 cm−1 decreased due to the allyl and the characteristic peaks of the oxazine ring respectively, which indicated that the ring-opening reaction and polymerization of the oxazine ring had taken place. Meanwhile, the peak at 3203 cm−1 was due to the phenolic hydroxyl group that occurred at 160 °C/1 h. With curing proceeding, the peak at 1224 cm−1 vanished because the epoxy group decreased, whereas the peak at 1012 cm−1 increased due to the ether bond. The reaction between the phenolic hydroxyl group and epoxy group was found. Therefore, these results proved that the copolymer had been completely polymerized [The thermally activated polymerization reaction of the copolymers was also studied by non-isothermal DSC, as shown in ant data , the curTGA in the nitrogen and air atmospheres, derivative thermograms, and tan δ curves of the nanocomposites are shown in Tg) of the nanocomposites was slightly lower when compared with the BOZ:E matrix. As the POSS cage structure consisting of Si atoms and O atoms was bulky, this hindered the formation of the benzoxazine/epoxy resin cross-linked network to a certain extent and reduced the crosslink density [2 yield that was produced by the thermal degradation of POSS. The char yield in the air atmosphere was far less than in N2. As can be seen from the thermograms, not only in the N2 but also in the air atmosphere was the weight loss curve of the BOZ:E matrix not significantly altered by the presence of SPOSS. Similar weight loss traces were found from the TGA profiles. The TGA clearly showed that the 5% mass loss temperature (T5%) of SPOSS-PBZ-E nanocomposites was slightly lower than the BOZ:E matrix except for the modified composites with a 20 wt % addition of SPOSS-BOZ in the N2 atmosphere Therefore, the decrease in T5% and Tg was consistent. At a later period of the degradation process, the degradation rate slowed down with the increased POSS content and the Tpeak of all the SPOSS-PBZ-E hybrid nanocomposites almost remained unchanged with respect to that of the BOZ:E matrix. The steric hindrance and interaction with the polymer chains caused by the incorporation of the POSS cages may result in a limit to the moving of polymer chains during the thermal degradation and lead to a small increase in thermal stability. Moreover, the thermal stability of the inorganic component (POSS) was very good and, during combustion, the POSS units could form a ceramic superficial layer because of the low surface energy of the siloxane structure of POSS [2 and 2.8% in air, respectively. From the DMA experimental data, the glass transition temperature , and cotton ignited by flaming particles or drops. The composites of PBZ-E without SPOSS-BOZ and 5% SPOSS-BOZ burned readily and had no rating; however, even with an incorporation of 10% of SPOSS, the burning behavior of blends altered and there was no dripping. The UL 94 V-1 rating was obtained and the total afterflame time (t1 + t2) was reduced in comparison to the 5% POSS-BOZ-E. During combustion, the SPOSS-BOZ nanocomposites formed a dense SiO2 layer that could prevent the penetration of combustion. When the POSS content cannot reach this level, an effective SiO2 layer will not be formed during combustion. The flame retardancies of the SPOSS-BOZ-E composites are presented in Cone calorimetry is used as a more promising technique to compare and evaluate the fire performances of polymeric materials. The fire performances of the composites were compared using time to ignition (TTI), peak heat release rate (pHRR), average HRR (aHRR), total heat release (THR), total smoke production (TSP), and total smoke release (TSR). These parameters are reported in TTI is used to determine the influence of flame retardant on ignitability. As shown in 2, and the pHRR values of the 5% SPOSS-PBZ-E, 10% SPOSS-PBZ-E, 15% SPOSS-PBZ-E, and 20% SPOSS-PBZ-E were 957, 962, 942, and 769 kW/m2, respectively. It is clear that SPOSS-BOZ apparently hinders the decomposition of the composites and improves the flame retardancy. The addition of 20 wt % SPOSS-BOZ caused reductions in the pHRR, aHRR, and THR values by about 33%, 27%, and 25%, respectively  was synthesized by the thiol-ene (click) reaction. The curing process of SPOSS/polybenzoxazine/epoxy has multiple polymerization mechanisms according to the oxazine ring-opening and epoxy resin (EP) polymerization, which was characterized by FTIR, and showed that the phenolic hydroxyl group was formed after the ring opening of the oxazine ring, which promotes the ring-opening polymerization of epoxy. The X-ray diffraction analysis and the SEM micrographs of the SPOSS-PBZ-E nanocomposites indicated that SPOSS was chemically incorporated into the hybrid nanocomposites in the size range of 80–200 nm. The thermal properties and the char yield of the nanocomposites increased, and the highest maximum rate of weight loss decreased with respect to the addition of the SPOSS-BOZ organic–inorganic hybrid. At the same time, the flame retardancy of nanocomposites was improved when compared with the pure benzoxazine/epoxy blend. When the amount of SPOSS reached 10 wt % or more, the UL 94 vertical burning rating reached V-1, and when the SPOSS content reached 20 wt %, both the thermal stability and the flame retardancy of the curing system were improved. In the cone calorimeter testing, the addition of SPOSS-BOZ hindered the decomposition of the composites and the addition of 20 wt % SPOSS-BOZ caused reductions in the pHRR, aHRR, and THR values of approximately 33%, 27%, and 25%, respectively. Then, the morphologies of the chars were also studied by SEM and EDX, which indicated that during the combustion process of the nanocomposites, the POSS nanoparticles accumulated on the surface and formed a ceramic layer in the exterior char residue.


The resulting 11-item scale mostly assesses appearance-related attitudes, fear of stigma, affect, and desire for change, all of which may be a natural response to societal weight stigma, even in the absence of self-devaluation. Items pertaining to self-blame, stigma awareness, perceived legitimacy of weight stigma, and most items pertaining to self-worth, were excluded from the final scale. It is unclear whether an a priori assumption of multi-dimensionality would have produced different results.Internalized weight stigma (IWS) is generally operationalized as self-devaluation due to weight in higher-weight individuals. The most commonly used measure of IWS, the Weight Bias Internalization Scale (WBIS), was developed from an original pool of 19 items. Item selection was guided by statistical techniques based upon an Exploratory and confirmatory factor analysis of the original 19-item questionnaire was conducted in 931 higher-weight individuals.A 13-item two-factor structure was identified. Factor 1 comprised seven items that could be loosely conceived as weight-related distress. Factor 2 comprised six items, all of which pertained to weight-related self-worth. Tested individually, the six items making up the self-devaluation factor were an excellent fit for the data on all fit indices.per se is the construct of interest.IWS is a multi-dimensional construct. The two-factor WBIS (WBIS-2F) provides options to explore the relationships between different aspects of IWS and upstream and downstream variables. The Self-Devaluation subscale is suitable for standalone use when weight-related self-devaluation  Weight stigma can be broadly defined as exposure to negative attitudes, behaviors, or structural indignities that befall higher-weight individuals because of their weight or size. Higher-weight individuals experience weight stigma in practically every domain of daily life . In addiand subsequently devaluing yourself because of it  who self-identified as “overweight,” “obese,” or “fat”The choice to use these three terms to describe weight status was a deliberate one. Higher-weight individuals have different preferences for the terminology used to describe their bodies, often finding one or more of the terms offensive. For example, members of the “size acceptance” community – one of the groups targeted in the recruitment process – prefer the word “fat” and dislike medicalized terms of body weight . Althoug2. Extensive evidence testifies to the fact that self-identification of body size is either an equally or more consistent predictor of cognitive, affective, and behavioral correlates than is objective BMI . The study was approved by the University of Birmingham Ethical Review Committee.The survey was conducted using a dedicated survey platformstrongly disagree) to 7 (strongly agree), with higher scores indicating greater weight-related self-stigma. Internal reliability of the WBIS-19 was 0.92. Participants were asked to provide age, gender, and ethnicity, and to report height and weight measurements, which were used to calculate BMI. The option to decline to answer any of these questions was provided.3Internalized weight stigma was measured with the WBIS-19  see Tab. Items wn = 2) or two (n = 1) items on the WBIS-19. Missing values analysis indicated no overall pattern of missingness, Little’s MCAR test χ2(75) = 91.7, p = 0.09, indicating that these data were missing completely at random. Independent samples t-tests confirmed no response differences between participants with or without BMI data available. As BMI was collected predominantly for descriptive purposes, and was not included in the hypothesized model, missing BMI values were not imputed. Given the very low prevalence of missing data on the WBIS-19, no imputation was used and factor analyses were conducted with listwise deletion. Missing values on demographic variables  were also not imputed.Fifty-one participants (5.5%) were missing height and/or weight information such that BMI could not be computed. Three participants (0.003%) had missing responses on one (N = 481), using principal axis factoring and direct oblimin rotation with Kaiser normalization. It was stipulated that item factor loadings >0.3 represented a substantive contribution of the item to a factor. Given the large sample size, factor extraction decisions were based on the scree plot, rather than eigenvalues  using maximum likelihood estimation. Model fit was assessed using χ2 values, comparative fit index (CFI), and standardized root-mean-squared residuals (SRMR). Cut-off values of 0.95 for the CFI and 0.08 for SRMR, respectively, are generally considered to indicate a relatively good fit of the hypothesized model to the observed data  and one was aged 17. These participants fell outside the age range specified in the approved ethical application for this study and were also excluded. The final sample size was therefore 931.A total of 1154 participants began the study and 963 (83.4%) completed it. Twenty-six participants (2.7%) had a BMI less than 25 kg/mM = 40.2, SD = 11.4; 3.8% missing), and BMI range was 25.0–95.0 kg/m2 . Further breakdown of BMI distribution indicated 14.1% had BMI between 25.0–29.9 kg/m2, 21.4% between 30.0–34.9 kg/m2, 17.9% between 35.0–39.9 kg/m2, 27.8% between 40.0–49.9 kg/m2, and 13.3% had BMI greater than or equal to 50.0 kg/m2. Just over one-third were living in the United Kingdom and just over a half in North America – no other region accounted for more than 5% of the sample. Participants were also highly educated, with three-quarters having a college degree or higher, and 61.3% listed their occupation as managerial, administrative, or professional; 9.5% were students, 5.2% unemployed, 20.9% other, 3.2% missing.The sample was predominantly female  and White . Age range was 18–69 years  with low skewness  and kurtosis , indicating a good distribution of IWS scores. Small but significant correlations were observed between WBIS-19 and BMI .WBIS-19 scores were normally distributed  suggested a three-factor structure for the WBIS-19, explaining 54.8% of the total variance (see F1) included 11 items and was almost identical to the standard WBIS-11. While these items appear conceptually diverse, this factor could be loosely conceived as “weight-related distress” – negative cognitive and affective states resulting from weight status, for whatever reason, whether related to how you look, how others treat you, if you blame yourself for getting that way, and so on. Corrected item-total correlations for the 11-item factor ranged from 0.55 to 0.81. The second factor (F2) initially comprised four items, two pertaining to the perceived legitimacy of anti-fat attitudes – “It really bothers me that people look down on overweight people” and “I believe that society’s prejudice against overweight people is unfair” , and two pertaining to self-blame – “It’s my fault that I’m overweight” and “If only I had more willpower, I wouldn’t be the weight I am” – that had similar factor loadings across both F1 and F2  comprised six items, all of which pertained to weight-related self-worth, and this factor was labeled “weight-related self-devaluation.” Corrected item-total correlations for this six-item factor ranged from 0.36 to 0.66. F1 and F3 correlated 0.530, but F2 did not correlate strongly with either of the other factors . Additionally, F2 contributed the least proportion of total variance explained .Exploratory factor analysis based on a random half (approximate) of the sample . In contrast, the items pertaining to self-blame correlated greater than 0.3 with 11 of the remaining 17 items. Given the imbalance of the number of items across the three factors, the low correlation of F2 with the other two factors, the very low correlations between items 14 and 18 and the remaining 17 items, and the relatively small contribution to the total variance explained, it was decided to delete the two items pertaining to perceived legitimacy but to retain items 1 and 6 at this stage. Thus, the analysis was re-run with the remaining 17 items.F1, the EFA was re-run pre-specifying a two-factor extraction. This analysis produced a clear pattern of factor loadings, with 11 items loading onto a weight-related distress factor (including the items pertaining to self-blame), and six onto a weight-related self-devaluation factor. Internal reliability of the weight-related distress factor was 0.926. Internal reliability of the weight-related self-devaluation factor was 0.768. Item 16 – “I feel that being an overweight person does not make me unworthy of a loving relationship” (reverse-scored) had a lower item-total correlation than the other items on the factor (0.360), and its deletion would have increased the internal reliability to 0.794. However, given the relatively small size of this improvement, that deletion would also have a large impact on scale variance – reducing it from 39.4 to 27.6, and the smaller number of items on this factor, a decision was made to retain this item at this stage.EFA of the WBIS-17 again produced three factors, explaining 56.0% of the total variance, with the items pertaining to self-blame no longer cross-loading, but now loading uniquely onto their own factor. However, as these items have previously loaded onto N = 450) tested the 17-item two-factor model identified by exploratory analysis. The factors were allowed to covary. The two-factor structure of the WBIS-17 was a poor fit to the data  indicated nine pairs of items with values above 10. The highest of these was for item 12 – “I am OK being the weight I am” (reverse-scored) and item 5 – “I wish I could drastically change my weight,” MI = 77.8. Item 5 had slightly higher estimated factor loading and estimate/standard error, and was slightly more strongly correlated with other items on the scale; thus, item 12 was deleted and the CFA re-run.Confirmatory factor analysis using the remainder of the sample  to excellent  fit for the data.7 By comparison, the standard unidimensional WBIS-11 was an acceptable  to poor (RMSEA) fit for the data.The resulting two-factor WBIS-13 (WBIS-2F) was a very good fit to the data. Three MIs had a value above 10, but none involved overlap on face validity, and no further changes were made. The final 13-item scale therefore included a seven-item weight-related distress factor and a six-item weight-related self-devaluation factor .6 AdditiM = 4.6, SD = 1.5) than weight-related self-devaluation . Weight-related distress and self-devaluation were moderately correlated . Cronbach’s α for the two subscales was 0.910 and 0.763, respectively.Using the full sample, scores on the two factors indicated higher levels of weight-related distress  was a good fit to the data. Exploring participants’ scale scores, the fact that scores on the weight-related distress subscale were notably higher than those on the self-devaluation subscale provides support for the contention that these items are measuring something different to self-devaluation. It could be that the weight-related self-devaluation subscale provides a true measure of perceived internal worthiness, or lack thereof, whereas the weight-related distress subscale represents feelings and thoughts associated with fears of not fitting into society. Thus, this multi-dimensional scale structure provides a more nuanced representation of internalized weight-related cognitions and affect than does the standard WBIS-11.The Weight-Related Self-Devaluation factor provided the best statistical fit to the data when tested individually, suggesting that these six items could be used as a standalone scale when the research question focuses specifically on weight-related self-worth. Although the Weight-Related Distress factor was a good fit for the data when tested independently, its similarity to the standard WBIS-11 may negate any benefit of using it in this way, and it may be preferable to continue use of the standard WBIS-11 when a broader conceptualization of weight-related self-stigma is of interest, in order to retain comparability with the extant literature. Additionally, a small number of items on the weight-related distress subscale do refer to self-worth. Future work on IWS may benefit from revisiting this construct, using a larger number of pool items, possibly generated with input from the target population, a large, diverse sample, and a thorough psychometric validation of the resulting scale(s).This study has a number of strengths. First, the large sample size permitted cross validation of the factor structure in two groups of non-treatment-seeking higher-weight individuals. Second, the participants represented a good range of body sizes across the higher-weight spectrum and diverse weight-related attitudes. However, there are also a number of limitations. The sample lacked gender, ethnic, and geographic diversity, which precluded testing of measurement invariance and latent mean differences on factor scores across groups. Subjects may have been prone to social desirability responding, particularly with regard to the perceived legitimacy questions. As no measure of social desirability responding was used, it was not possible to test this. Additionally, if the WBIS-2F and its subscales are to be used in future research, further assessment of the psychometric properties of the scale(s) will be needed.Internalized weight stigma may usefully be conceptualized as a multi-dimensional construct, encompassing both weight-related self-devaluation and more generalized cognitions and emotions related to living in a high-weight body in an anti-fat environment. The two-factor WBIS-2F could be used to explore the relationship between specific aspects of weight-related self-stigma and other upstream and downstream variables. Additionally, the six-item Self-Devaluation subscale aligns most closely with the original conceptualization of IWS, as a measure of reduced weight-related self-worth. This scale is suitable for standalone use when self-devaluation is the construct of interest.This study was carried out with written informed consent from all subjects in accordance with the Declaration of Helsinki. The protocol was approved by the University of Birmingham Ethical Review Committee.AM conceived the study and was responsible for data acquisition and analysis. AM and SH contributed to the design of the study and interpretation of the data. AM drafted the initial version of the manuscript. Both authors were involved in critical revision of the manuscript, approved the final manuscript, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Responding to emergency alarms is a daily occurrence for personnel in safety-critical occupations, and is associated with negative health outcomes in this population. The purpose of the present study was to determine the acute inflammatory response to an isolated emergency alarm mobilisation in both day and night conditions.Sixteen healthy males (mean age 25 ± 4 years) spent four days and nights in a sleep laboratory and were required to mobilise to an emergency alarm either during the day (1558 h), or from nocturnal sleep (0358 h). Pro  and anti-inflammatory (IL-4 and IL-10) cytokine responses to each alarm mobilisation were compared to time-matched control conditions without the alarm and mobilisation stimulus.Analysis revealed no significant drift of cytokine levels at 1400 h across the study (P≥0.139). The plasma concentration of anti-inflammatory cytokine IL-4 was 84% greater in the 2-h sampling period following night alarm mobilisation compared to a night control of gentle awakening (P = 0.049), no other condition-by-time interactions were observed. The majority of inflammatory concentrations did not significantly change between alarm mobilisation and control conditions, in either day or night trials.These findings may reflect the lack of a true emergency (and the perceived stress) for the alarm mobilisation, together with the neutralising effect of different circadian biorhythms on inflammatory cytokine concentrations. Emergency alarms are ubiquitous in clinical environments, industry, and safety-critical occupations –3, with -1 [The exact mechanism for an elevated risk of CVD-related incidents during an alarm mobilisation is yet to be elucidated, however alarm response has been acutely shown to increase heart rate by up to 66 beats·min-1 , 12, and-1 –13. Fire-1 , 15. Thi-1 , 17, whi-1 , 19 and -1 . Meta-an-1 , 22. How-1  may be a-1 , 23.h2) [Of further consideration is the potential for alarms to disrupt and restrict sleep in on-duty personnel who are required to respond to emergency alarms. It is unknown as to whether sleep disruption instigated by a night-time alarm mobilisation produces an acute inflammatory response. Moreover, mixed findings have been reported in the few published studies that have examined the acute response of cytokines after abrupt waking and disrupted night-time sleep in the second half of the night  , 25. Strh2) , to fosth2) . Howeverh2) .Therefore, gaining an understanding of the acute inflammatory response to a single alarm is the first necessary step in elucidating any longer-term relationship between an alarm stress response and CVD-related mortality in emergency personnel , 9. Furt-2, were recruited for this study. Males were recruited as they make up the vast majority of all emergency service personnel , body mass; 71.9 ± 7.7 kg, and body mass index; 23.1 ± 2.2 kg·ml , and mal , was presented outside the participants’ individual rooms at 1558 h on Day 1, for a maximum duration of 2 min. This alarm intensity is consistent with the literature and occupational practice –13, 37. No alarms were presented during the afternoon in either Day 3 or 4, despite participants expecting an alarm at any time of the day. On the third night, participants received one of two conditions at 0358 h, an alarm stimulus requiring mobilisation to the testing station , or a gentle awakening (control) condition. This time was chosen to contextualise the circadian nature of cytokines. In the night alarm condition, an alarm was presented outside participant’s rooms, as per the day alarm condition. Upon alarm sounding, lights were brought up to their full level, and participants then followed the same protocol as the day alarm mobilisation. Participants were exposed to the alternate condition on the fourth night. In the night control condition incorporating gentle awakening, lights in participant rooms were activated externally but only brought up to a half level, after which researchers quietly knocked and entered to provide verbal commands. Participants remained in their beds but slowly sat up to provide the T0 finger prick blood sample. Following this they walked slowly to desks positioned in their rooms to provide all other samples. The same collection time points were used for sampling in both day and night conditions. In both night-time test conditions, subjects did not return to sleep after data collection (0400–0600 h), and therefore a cross-over design was employed  to counterbalance the potential pro- or anti-inflammatory effects of sleep restriction.Dietary intake was controlled throughout the study, and no food was consumed during the 90 min prior to blood collection. In an attempt to further regulate emotion, alertness and stress levels, no caffeinated beverages were allowed for the duration of the study. Similarly, participants spent the majority of their time in individual bed-sit style rooms, and did not have access to natural light for the duration of the study due to the design of the sleep laboratory. Physical exertion was not permitted, however participants were encouraged to perform light stretching if they reported joint or muscle stiffness.2EDTA , and immediately stored on ice until the end of the sampling period. Samples were then centrifuged at 5,000 rev·min-1  for 10 min to separate plasma, and stored at -80°C until analysis. Finger prick blood sampling was chosen to avoid the blood coagulation effects, and immune challenges produced in samples obtained via venupuncture and intravenous cannulation [Finger prick blood samples were collected using BD-microtainer retractable lancets . Samples (≥375 μL) were collected in a 500-μL micro-test-tube coated with Knulation .TM MAP Human High-Sensitivity Cytokine kit . Each assay was performed strictly according to the manufacturer’s protocol for plasma samples. The mean inter-assay coefficient of variation was 9.5% and the mean intra-assay coefficient of variation was 9.3%.Inflammatory cytokines IL-4, IL-6, IL-1β, TNF-α, IL-10 and IL-8 were analysed simultaneously using a 6-plex MilliplexDue to the larger aims of the project, and a lack of research regarding inflammatory response to an alarm, a priori power calculations were based on changes in heart rate in response to an emergency alarm . From thStatistical analyses for cytokine samples were completed using STATA statistical software release 15.0 . Cytokine data were assessed for normality using the Kolmogorov-Smirnov Test which is suitable for small samples . Due to The mean baseline plasma cytokine concentrations collected at 1400 h on the day of each condition are shown in This was due to a 77% decrease in concentration at T90 compared to T0 in the day alarm condition (P = 0.014), and a 9% decrease in the day control condition that did not reach statistical significance  in IL-4 at T120 compared to T0 in the night alarm condition, compared to night inactive (condition-by-time interaction P = 0.049). This was due to a 43% increase in concentration at T120 compared to T0 in the night alarm condition (P = 0.168), and a 41% decrease in the night inactive condition , 37% higher than T90 (P = 0.020), 37% higher than T120 (P = 0.036), and 43% higher than baseline levels taken at 1400 h . FurtherThe overarching aims of this study were to determine whether day and night alarm mobilisations could be classified as acute immunological stressors. Changes in the plasma concentration of anti-inflammatory cytokine IL-4 were higher in the 2-h sampling period following night alarm mobilisation compared to a gentle awakening control condition (P = 0.049), however no between-group differences over time were seen for any cytokine following day alarm mobilization, compared to the day control condition. In light of these findings, it is unclear whether a single alarm mobilisation can be classified as an acute immunological stressor.r = 0.66, P = 0.028). The current data therefore implies that an alarm mobilisation in a non-emergency situation, using ‘healthy normal’ participants, may not be capable of provoking a pro-inflammatory response, despite its ability to activate the SAM system [A relationship between sympathetic activation and immune response has been previously reported, with studies demonstrating correlations between acute changes in heart rate and inflammatory markers , 44. FurM system .To our knowledge, there are no previous studies that have evaluated changes in circulating inflammatory markers following alarm mobilisation. In the current study, there are a number of factors which may explain why we observed no between-condition changes in the various inflammatory markers relative to control conditions, including an inadequate exposure to the psychological stressor, and the nature of the stressor used in our protocol. An initial explanation for our lack of between-condition change in the pro-inflammatory markers in day or night conditions relates to controllability, in that the laboratory-based alarm stimulus was not perceived by the participant to be a serious stressor and subsequently failed to elevate inflammatory levels . StudiesIt is established that acute laboratory stress is associated with an increase in circulating and stimulated concentrations of pro-inflammatory biomarkers , 22, butIn contrast to our findings for IL-1β, concentrations of IL-6 and TNF-α demonstrated significant increases two hours after day alarm mobilisation (65% and 17% respectively), compared to their relative concentrations at alarm onset (T0). However, these were not different to concentrations in the control condition. Meta-analytic data has previously reported increases of IL-6 and TNF-α in response to a range of acute psychosocial stress tasks and naturalistic stressors, typically between 45–120 min after exposure to the stressor , 22. In Pertinent to emergency service personnel, elevated levels of circulating IL-6 have been linked to the increased synthesis of C-reactive protein, with both of these markers implicated as predictors of myocardial infarction and arterial disease in healthy men , 55. AltAt the onset of night alarm mobilisation, concentrations of the anti-inflammatory cytokine IL-4 underwent a significantly greater decrease from baseline, compared to the night control condition , before Significant elevations of the anti-inflammatory cytokine IL-4 have been previously witnessed as a result of surgical stress , which wContrary to our hypothesis, pro-inflammatory cytokine concentrations did not increase following a night alarm mobilisation compared to the control condition. There is evidence that peak production and secretion of the pro-inflammatory cytokines occurs during nocturnal sleep (0000–0500 h) , 29, andThere were several strengths to this study, most notably it is the first trial to investigate the inflammatory response to an acute alarm mobilisation, which may be viewed in part as a stressful auditory and physical stressor, during periods of day and night. Furthermore, the use of within-participant control conditions attempted to counteract the individual and circadian variability of circulating cytokine concentrations , 28. Futper se may constitute a definitive immunological stressor. Future research may consider whether an acute inflammatory response may be elicited with repeated alarms, alarm mobilisations presented at different times during a circadian cycle or in actual emergency conditions. With this information, any injurious properties of habitual alarm mobilisation may be better understood.In summary, a night alarm mobilisation was associated with greater increases in IL-4 concentration across a 2-h sampling period, in comparison to gentle awakening. However, a day alarm mobilisation did not produce an inflammatory response in comparison to a relative control condition. No other significant between-condition changes over time were observed for any other cytokine, and there was no drift of cytokine levels at 1400 h across the study. The current findings suggest a lack of response of inflammatory markers to alarm mobilisation, potentially mediated by circadian, physiological and psychological mechanisms. Currently, it cannot be determined whether an alarm mobilisation


High content of lycopene was detected in red tomatoes. The orange tomatoes contained three dominant carotenoids, namely δ-carotene, β-carotene, and prolycopene. The yellow tomatoes showed low levels of carotenoids compared to red or orange tomatoes. Based on the HPLC profiles, genes responsible for overproducing δ-carotene and prolycopene were described as lycopene ε-cyclase and carotenoid isomerase, respectively. Subsequent genetic analysis using DNA markers for segregating population and germplasms were conducted to confirm the hypothesis. This study establishes the usefulness of metabolic profiling for inferring the genetic determinants of fruit color.Carotenoids are essential for plant and animal nutrition, and are important factors in the variation of pigmentation in fruits, leaves, and flowers. Tomato is a model crop for studying the biology and biotechnology of fleshy fruits, particularly for understanding carotenoid biosynthesis. In commercial tomato cultivars and germplasms, visual phenotyping of the colors of ripe fruits can be done easily. However, subsequent analysis of metabolic profiling is necessary for hypothesizing genetic factors prior to performing time-consuming genetic analysis. We used high performance liquid chromatography (HPLC), employing a C Solanum lycopersicum L.) is a model crop for studying the biology of fleshy fruits, especially carotenoid biosynthesis [Tomato (ynthesis . In planynthesis . The briynthesis . Dietaryynthesis . Each caynthesis . Similarynthesis . Therefocis-lycopene. Carotenoid isomerase (CRTISO) converts cis-lycopene to trans-lycopene by isomerization reactions at 7, 9 and 7′, 9′ cis-bonds. The trans-lycopene is cyclized through two routes involving lycopene ε-cyclase (LCY-E) and lycopene β-cyclase (LCY-B). The LCY-E and LCY-B catalyze the synthesis of δ-carotene and α-carotene, respectively, through one of the routes that branches at trans-lycopene and leads to the formation of lutein. The other route leads to the synthesis of γ-carotene and β-carotene by the catalysis of LCY-B and ultimately results in the formation of neoxanthin.During ripening, the color of tomato fruit changes from green to red because of increased synthesis of carotenoids, especially lycopene. The carotenoid biosynthesis pathway was identified using genetic and biochemical approaches  7,8,9,1,17,18,192 concentration, temperature, photo-oxidative stress, and endogenous signals, and the biosynthetic genes are spatio-temporally regulated at the transcriptional level [yellow flesh (r) mutant (with loss of PSY1 function) has yellow ripe fruits because of the lack of carotenoid synthesis during ripening [tangerine (t) mutant (with loss of CRTISO function) rarely produces trans-lycopene [Delta  and Beta  mutants synthesize high amounts of δ-carotene and β-carotene, respectively, at the expense of trans-lycopene [t, Del, and B mutants show orange ripe fruits because of differences in the carotenoid composition compared to that in the red tomatoes.The accumulation of carotenoids is influenced by several factors, such as the intensity of radiation, COal level ,29,30,31al level . The yellycopene . The Dellycopene ,16. The http://tgrc.ucdavis.edu/). It is necessary to classify the newly developed resources along with the previously identified resources. The classical phenotyping might not be able to resolve the genetic variations that show similar fruit colors, and time-consuming genetic analysis is impractical. However, prior to the genetic analysis, metabolite analysis can easily and quickly provide information for investigations on the genes involved in carotenoid accumulation in tomato fruits [30 stationary phase column and are identified by determining the absorption spectrum with a photodiode array detector (PDA) because different carotenoids have unique structures and different absorption maxima [The tomato mutants described above were identified by positional cloning and transgenic approaches, which are time-consuming. Several such mutants have been developed ,33 and go fruits . Direct o fruits . Changeso fruits . Thus, ao fruits . Among to fruits ,39,40. Cn maxima . In thisS. lycopersicum ‘LA3475’  [apricot (at) [at mutant, the flower color showed deficiency of carotenoids unlike in the r mutant. The flower color in Sugar Yellow was same as that in the wild type (data not shown) suggesting that the at mutation may not affect the fruit color variation in Sugar Yellow. The ripening mutants normally showed lack of carotenoid in addition to the ripening phenotypes, such as higher fruit firmness. Sugar Yellow showed normal ripening patterns as in the wild type.The HPLC profile in Sugar Yellow tomato with yellow-colored fruits was different from that of the other tomato lines. The β-carotene content was much lower than that of other lines and the level of other carotenoids were also very low. Furthermore, lycopene was not detected D. These lesh (r)  or apriccot (at) , showingcis-lycopene), showed different absorption spectrum and retention time compared to cis-lycopene detected in red tomato. Thus, prolycopene is a representative carotenoid only present in these lines. Subsequently, these lines were assumed to be regulated by the mutation in CRTISO [cis- to trans-lycopene isomerization. Therefore, interrupting the synthesis of trans-lycopene may lead to the accumulation of its precursor, prolycopene, and the appearance of orange color.LA0030, LA3183, LA3533, LA3682, Gold Minichal, and Cispene with orange ripe fruit color contain lower amounts of β-carotene and lycopene compared to that in LA3475. HPLC analysis of these lines showed major absorption peaks at 450 nm at a retention time of 20.6 min, which were identified to be prolycopene based on the absorption maxima and spectrum E. Prolycn CRTISO , which itrans-lycopene compared to the amounts of these present in the red tomato fruit of LA3475  and red  colored ripe fruits at a ratio of 3:1 . The genotypes of LCY-E in the F2 population were analyzed using the INDEL marker. We could identify 19 homozygous individuals of the LA4099 type (orange fruits), 47 heterozygous individuals (orange fruits), 28 homozygous individuals of the LA3475 type (red fruits), as per the expected genotypic ratio of 1:2:1 . The genotypic data co-segregated with the phenotypic data, suggesting that LCY-E is the genetic determinant of the dark orange fruit color in the δ-carotene overproducer, LA4099.The dark orange colored fruit in LA4099 contained a high amount of δ-carotene and a low amount of f LA3475 A. Based rt codon A. To ideeveloped . To veripulation B. The F1LCY-E, is a candidate source for increased lutein. In a previous study, transgenic tomato over-expressing LCY-E showed higher accumulation of δ-carotene. This transgenic line was crossed with the Beta mutant (overexpressing LCY-B) and the resulting hybrid line was shown to accumulate high amounts of lutein because of the over-expression of LCY-E and LCY-B [The δ-carotene over accumulating tomato, with high activity of nd LCY-B . Lutein,nd LCY-B . Therefotrans-lycopene compared to LA3475  did not contain the deletion  showing orange fruit color accumulated increased amounts of prolycopene and reduced amounts of o LA3475 E. Becausdeletion , Table 2cis-lycopene isomers mainly present in the human blood and tissues although trans-lycopene is the principal form of lycopene in red tomato [cis-lycopene isomer from the t mutant tomato sauce compared to equal amounts of trans-lycopene from red tomato sauce in their total serum [CRTISO mutants would be useful resources for improving the bioavailability of lycopene [Prolycopene is one of the d tomato . Humans al serum . Thus, Clycopene .trans-lycopene, whereas carotenes and xanthophylls impart orange and yellow colors, respectively. Thus, it is difficult to distinguish the genetic variation visually because most of the tomato mutants with defect in the carotenoid biosynthesis genes show orange and yellow colored ripe fruits. In this study, we identified distinct carotenoid profiles of tomatoes showing diverse fruit colors and inferred the candidate genes for each variation in addition to the genetic analysis. Visual phenotypes were often connected with dramatic metabolic changes. Thus, metabolic profiling can be a highly efficient method to infer the genetic determinants regulating the metabolite synthesis or accumulation before conducting the time-consuming genetic analysis.The red color of tomato is because of the higher accumulation of Thirteen tomato germplasms and three commercial cultivars  were used for the profiling of carotenoids. The tomato plants were grown in a greenhouse with 16 h light/8 h dark period at temperatures ranging from 19 to 28 °C, 50–70% relative humidity. After fruit setting, 2 g of water soluble NPK fertilizer  per each plant were supplied every week. Three ripe fruits (ten days post-breaker stage) per each line were harvested for evaluation of fruit colors and extraction of carotenoids from the pericarp. The fruit colors were evaluated by three persons. The harvested pericarps were immediately frozen in liquid nitrogen and stored at −80 °C before the extraction of carotenoids.To prevent the degradation and oxidation of carotenoids, all the experiments were conducted under limited light conditions. For carotenoid extraction, the method of extraction described by Vrebalov et al.  was usedt-butyl ether and 242 μL of MeOH. The carotenoid suspension was filtered through a 0.45-μm syringe filter  and put in an HPLC vial. The carotenoid analysis was performed using a 1260 Infinity series HPLC instrument  and the Chemstation software. The injection volume was 25 μL and the flow rate was 1 mL min−1. The column temperature was maintained at 25 °C for all the analyses. The carotenoids were separated under a polar to nonpolar gradient using mobile phases consisting of 100% methanol with 0.1% ammonium acetate (A) and methyl t-butyl ether (B) through a guard column  and C30 stationary phase . A gradient elution was performed with 100% of A for 6 min, which was then gradated to 4% of A and 96% of B over 20 min. After 26 min, A was increased to 100% until 36 min. The elution of carotenoids was observed on an online Diode Array Detector (PDA) at five wavelengths . The carotenoids were identified based on their absorption maxima and spectrum [The carotenoid extracts were suspended in 250 μL of methyl spectrum ,41.l-ascorbic acid in a 2 mL tube. The extract was incubated at 65 °C for 15 min. Thereafter, 600 μL of chloroform:isoamyl alcohol (24:1 v/v) was added and mixed well. The extract was centrifuged at 13,000 rpm for 15 min at 4 °C. The upper phase was transferred to a new tube and 500 μL of isopropanol was added and then the mixture was centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was removed and 70% ethanol was added to the pellet and then centrifuged at 13,000 rpm for 5 min at 4 °C. Ethanol was removed and the extract was centrifuged for 1 min. The remaining ethanol was removed and 30 μL of ddH2O containing 1% RNase was added to the pellet.The genomic DNA of tomato was extracted using a modified cetyltrimethylammonium bromide (CTAB) method . The youe-Taq DNA polymerase  in a T-100 Thermal Cycler , with 100 ng of each DNA template and 10 pmol of gene specific primers for LCY-E (Solyc12g008980) and CRTISO (Solyc10g081650) (E. coli (DH5α). The plasmid DNA was isolated by a plasmid prep kit . The sequence analysis was performed at SolGent. The LCY-e1 and CRTISO primers were used as DNA markers for screening the genotype of LCY-E and CRTISO, respectively. The PCR products obtained using the DNA markers were separated by electrophoresis on 1% agarose gel and were visualized by staining with ethidium bromide.Polymerase chain reaction (PCR) was conducted using g081650) . The PCRTomato fruits show various colors, such as red, pink, orange, yellow, brown, and purple. Among the diverse pigments affecting the fruit color, carotenoids are the primary determinants. Therefore, diverse color mutants often affect the carotenoid synthesis. Identification of genetic factors affecting the variation in fruit colors is time-consuming and labor intensive. Visual phenotypes underlying different genotypes are often indistinguishable from each other. Our study could distinguish similar fruit color variation by carotenoid profiling. Four distinct carotenoid profiles were investigated, and two candidate genes affecting fruit color and carotenoid variation were verified. Metabolite analysis using HPLC is an efficient tool for identifying the genes regulating the carotenoid biosynthesis in tomato. Metabolomics analysis in the field of genetics and breeding should be conducted for hypothesizing the mode of genetic action.


Whole-genome sequencing was used to identify potential causative mutations for a  col-4 allele of Neurospora crassa.In many cases, genes for commonly used genetic markers in model organisms have not been identified; therefore, it is of interest to identify the causative genes. Whole-genome sequencing was used to identify potential causative mutations for a  A single allele, Y152M43;30(r) 70007, was isolated in 1949  for col-4 is the major goal of this project.The low cost of genomic sequencing has made it feasible to bypass traditional genetics-based methods of gene identification and to approach this directly by sequencing the entire genome of interest. This has become very common in model systems with small genomes but is now being applied on a much larger scale to larger genomes  in various cancer genome projects. The N. crassa strains FGSC 3017  and FGSC 2489 (mating type A) were obtained from the Fungal Genetics Stock Center   was used for further analysis; mtr , on LG IV ∼1 map unit from col-4, has been well characterized, but this mtr allele has not. Similarly, the his-2 allele (encoding ATP phosphoribosylpyrophosphate pyrophosphorylase) on LG IR has not been characterized. DNA was isolated from N. crassa TR1 (N. crassa). FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used for quality control. No quality trimming or adapter trimming was found to be necessary. For single-nucleotide polymorphism (SNP)/indel identification, two separate subsets of 20 million reads from each paired-end fastq file were loaded onto the Galaxy analysis platform  (N. crassa reference sequences (build 10) for LG I and LG IV in fasta format were downloaded from the Broad Institute website  and loaded onto the Galaxy platform. Reads were aligned to each reference sequence with BWA 0.6.0 , a T-to-C transition upstream of locus NCU16770 , and an −/A indel between locus NCU06628 and locus NCU06629 ; all are intergenic and are likely to be regulatory. These were the only sequence differences from the 74A reference between the mtr gene and the beginning of a stretch of sequence from the Mauriceville background, which demarcates the location within which col-4 must be found. Additionally, single candidate mutations within the open reading frames of the his-2 and mtr genes were identified, i.e., a G-to-C transversion causing Ala to Pro (A32P) and a C-to-G transversion causing Tyr290 to the TAG stop codon, respectively.axy.org) . The N. PRJNA548559.The original sequencing data have been deposited in GenBank with BioProject accession number


Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from  Although the function of aquaporins (AQPs) as water channel proteins has been well studied for decades, it is a challenge to produce sufficient amounts of high-quality protein samples for applications such as creating bio-membranes for water filtration. Recent advances in material science have resulted in the creation of a variety of block co-polymers that are useful as artificial membranes for the integration of functional AQPs ,2. Howevl-CFPS mode) can promote the co-translational insertion of AQPs into the provided membranes [Previous studies have shown that high amounts of functional AQPs from both prokaryotic  and eukaembranes .E. coli and NtPIP2;1 from Nicotiana tabacum, were used as model proteins to investigate the process of their co-translational integration into liposomes. Improved homogeneity of the resulting aquaporin proteoliposomes was achieved via an interim detergent solubilization step. A shifted green fluorescence protein (sGFP) fused to the C-terminal of AqpZ was used as a monitor to follow the process of integration via confocal fluorescence microscopy. The function of water permeability was determined via stopped-flow spectrophotometry. The obtained results indicate a comparable functional incorporation of both aquaporins to conventional reconstitution methods. We demonstrate the functional insertion of both selected model AQPs from different organisms and present a simplified and streamlined protocol with improved efficiency.In this paper, the AQPs, AqpZ from NtPIP2;1 was amplified from yeast expression vectors created previously [NtPIP2;1. AqpZ-sGFP was constructed as reported before [8-tagged GFP. DNA templates used for CFPS were transformed into E. coli strain DH5a and isolated by standard plasmid purification kits .cDNA sequence of eviously  using thd before  and exprE. coli A19 extract. The preparation of S30 extracts, T7 polymerase, and basic compounds was prepared as reported before [® S20, a final concentration of 1% (w/v) was introduced into both RM and FM, substituting the extra volume of water. For CFPS in the presence of lipids (l-CFPS), preformed liposomes were added into the RM with corresponding final concentrations, substituting the volume of water.The CFPS system was constructed using a self-prepared d before ,16,17. Al-α-Phosphatidylcholine from soybean and detergent used in this study were purchased from Sigma-Aldrich . Lipid mixtures solubilized in chloroform were first transferred to a round-bottom flask. A thin lipid film was formed by rotary evaporation under a controlled vacuum. The flask was then placed in a vacuum chamber overnight. The lipids were rehydrated in 1 mL of assay buffer A  to a final concentration of 40 mg/mL. The mixture was vortexed for 15 min to form multilamellar vesicles. The multilamellar vesicle solution was passed at least 21 times through an Avanti Polar Lipids  mini extruder holding a 200-nm Whatman polycarbonate membrane filter  sandwiched with two filter supports on each side. The resulting unilamellar liposome solution was used for expression or water permeability experiments.AqpZ-sGFP was quantified by fluorescence measurement with excitation wavelength of 484 nm and emission wavelength of 510 nm . Furtherl-α-Phosphatidylcholine from soybean was performed as previously described [v/w) Triton X-100 , 20 mg/mL preformed liposomes , and 200 µg/mL purified AqpZ-sGFP. The reconstitution mixture was incubated at room temperature with gentle shaking for 30 min. The detergent was removed by SM-2 beads  according to the manual. Finally, the liquid reconstitution mixture was sent to ultracentrifugation at 500,000× g for 45 min. Then, the pellet was washed again with reconstitution buffer. After the wash step, the proteo-liposome solution was ultra-centrifugated again and finally re-suspended in 1.6 mL reconstitution buffer. Samples from the CFPS reaction were treated following the steps described below:In vitro reconstitution of purified AqpZ-sGFP into liposomes composed of escribed ,12, withg for 20 min. The pellet fraction containing precipitated protein, and protein inserted into liposomes and fused liposomes were collected. The supernatant, which did not contain any expressed protein, and a small amount of lipids was discarded. Pellet fractions were re-suspended in assay buffer A. In order to separate the precipitated proteins from proteins that were inserted and associated with lipids, detergents  or 1% (w/v) Octyl β-d-glucopyranoside (β-OG)) per 4 mg/mL of lipids were used to solubilize the pellet of lipids; the precipitated protein remained insoluble. After detergent re-solubilization, the sample was centrifuged again at 18,000× g for 10 min. The supernatant was collected and subjected to either dialysis or bio-beads treatment for detergent removal. For dialysis, a stepwise process was applied with 0.5%, 0.25%, 0.125%, and 0% β-OG final concentration in assay buffer A. Each dialysis was for around 6 h at 4 °C, with a final additional dialysis step for 12 h to completely remove the detergent. For the bio-beads treatment, 40 mg bio-bead per 1 mg/mL lipids reaction was applied, and the bio-beads were pre-incubated with 4 mg/mL liposome in assay buffer A incubation overnight. The proteo-liposome was reformed after removal of the detergent. All the samples with different treatments were extruded through a 200-nm membrane filter before application of the stopped-flow measurements.After overnight expression, the mixture was pelleted down via centrifugation at 18,000× 3 10%. The lipid-to-glucose molar ratio was 1:10. The mixture was treated with three cycles of freeze/thaw: 1 min freezing in liquid N2 followed by 5 min thawing at 42 °C (water bath), with vortexing between cycles. The sample was subsequently put on a siliconized glass slide and dried under the hood for several hours. In the next step, dried lipid film was covered with 25 µL MilliQ water and made to sit overnight in a humid closed petri dish (by putting pieces of wet paper tissue under the glass slide). The samples were collected and transferred into a new tube the following day.The formation of giant liposomes for confocal fluorescence microscopy was performed by following a modified protocol based on a description by Tsumoto et al. . In briek) was determined by the fitted curve of 6–10 measurements. The water permeability factor (Pf) of the vesicle samples was calculated using the equation described previously [S/0V is the vesicle’s initial surface-to-volume ratio, wV is the partial molar volume of water (18 cm3/mol), and S/V0 was calculated by determining the diameters of the proteo-liposomes using dynamic light scattering (ZetaPlus particle sizing software 2.27). The The setup of stopped-flow spectrophotometry was done as per a previous report . In brieeviously ,12,19: was used to stain the lipid molecules. In brief, 5 µL of the sample, 4 µL of the DABCO solution in water (20 mg/mL), and 1 µL of the Nile Red solution in DMSO (1%) were mixed together and incubated for 30 min. The specimens were covered by High Precision Microscope Cover Glasses  and sealed on four sides with nail polish.Confocal images were acquired using a TCS SP5 II (Leica) confocal laser scanning microscope equipped with a 100/1.4NA oil immersion objective. sGFP, Nile red was excited at 488, 561 nm laser. All images were recorded in room temperature using Image Processing Leica Confocal and Fiji .l-CFPS reaction conditions were used according to the previous description [2+, K+ and plasmid concentrations. The fusion construct of AqpZ-sGFP was expressed in the presence of liposomes (l-α-Phosphatidylcholine from soybean) with final concentrations up to 8.6 mg lipid per ml reaction mix (RM). After overnight incubation at 30 °C, the fluorescence of the RM was measured in order to determine the formed AqpZ-sGFP proteo-liposomes. Empty reactions without a plasmid template at a lipid concentration of 8.6 mg/mL were used as the negative control. CFPS of AqpZ-sGFP in the presence of detergent (d-CFPS) with final concentrations of 1% Brij® S20 was used as positive control. Within the analyzed range, the fluorescence signal was in a linear correlation with the lipid concentration  with the liposomes after overnight incubation. In order to monitor the distribution of AqpZ-sGFP, the liposomes were stained with Nile red, as described in the Methods section. In brief, the green channel showed the signal from sGFP, while the red channel showed the signal from Nile Red. The overlay of both channels indicates the distribution of AqpZ-sGFP in the liposomes. The liposome pellet obtained directly after the expression was inhomogeneous with randomly incorporated AqpZ-sGFP . Large cl-CFPS produced sample, a detergent solubilization step was introduced. Two commonly used detergents, Triton™X-100 and Octyl-β-d-glucopyranoside (β-OG), were selected to solubilize the pellet fraction. The final concentration of 0.42% Triton™X-100 and 1% β-OG for 4 mg/mL l-α-phosphatidylcholine (PC) were used (See Pf) of 150.60 ± 9.33 μm/s and 146.61 ± 6.03 μm/s, increasing to more than three folds if compared to the Pf value of 44.88 ± 1.33 μm/s for empty liposomes   see . Furtheromes See .Via fusion with the sGFP label, the insertion of aquaporin could be monitored via confocal microscopy. As shown in E. coli [Xenopus oocyte [In previous studies, the heterogeneous expression of recombinant AQPs using different expression systems was found to be successful, i.e., E. coli , yeast [E. coli , Xenopuss oocyte , and inss oocyte . Especias oocyte ,9,10,26.s oocyte . Howevers oocyte . Insteads oocyte . In this2+ ions and PEGs in the micro-environment of the CFPS system, both of which were routinely used for the fusion of liposomes [l-CFPS-produced GPCRs in previous reports [By applying liposome into the CFPS reaction mixture, pellet fractions were formed after overnight incubation. Previous research using a batch CFPS system was able to obtain AqpZ proteo-liposome with increased water permeability using slow centrifugation, followed by a dialysis step . Howeveriposomes ,30. As i reports . As showIn order to reform homogenous proteo-liposomes, a step of re-solubilization via detergent was then introduced. According to previous research, membrane proteins expressed in the CFPS system as precipitate can be selectively resolubilized with certain detergents . In contNtPIP2;1 proteo-liposome had a much higher value, when compared to that of the AqpZ-sGFP proteo-liposome. This might result from the influence of the fused sGFP, disrupting the formation of the stable tetramer [Quite a number of detergents like Triton™X-100, DDM, DM, and β-OG, among others, were used for membrane protein reconstitution . Previoutetramer .With a streamlined protocol to express and integrate aquaporins into proteo-liposomes, functional analysis of water permeability could become more robust, which certainly will boost downstream applications such as manufacturing of biomimetic water filtration membranes ,39, as wl-CFPS mode demonstrated a fast method for functional analysis of aquaporins. The key stage of detergent re-solubilization of the l-CFPS pellet fraction provides a single-step separation of precipitated and incorporated aquaporin. The results from both confocal microscopy image and stopped-flow spectrophotometry show the successful and functional incorporations of two model aquaporins into homogenous LUVs. Flexibility in the selection of detergents, lipids, and reforming proteo-liposomes could enable further study of other membrane proteins in various lipid environments.The present modified approach to create proteo-liposomes with functional aquaporins using the


After inserting a thin p-AlGaN layer into the n-AlGaN electron supplier layer, a conduction band barrier can be generated in the n-type electron supplier layer, which enables the modulation of the lateral current distribution in the p-type hole supplier layer for DUV LEDs. Additionally, according to our studies, the Mg doping concentration, the thickness, the AlN composition for the p-AlGaN insertion layer and the NPN-AlGaN junction number are found to have a great influence on the current spreading effect. A properly designed NPN-AlGaN current spreading layer can improve the optical output power, external quantum efficiency (EQE), and the wall-plug efficiency (WPE) for DUV LEDs.In this report, we locally modulate the doping type in the  Owing to various applications such as disinfection, water purification, medical treatment, and high-density optical recording –8, intenTherefore, at the current stage, research attention is laid on the p-side to facilitate the current spreading for DUV LEDs. In this work, different than other approaches, we propose and prove that the improved current distribution in the p-type hole supplier layer for DUV LEDs can be achieved by engineering the n-AlGaN electron supplier layer. The energy barrier is generated in the conduction band by modulating the doping type in the electron supplier layer, i.e., the n-AlGaN/p-AlGaN/n-AlGaN (NPN-AlGaN) structure is proposed and parametrically studied. Our results show that the lateral distribution for the holes can be homogenized by using the NPN-AlGaN junction, which therefore enhances the optical output power, the external quantum efficiency, and the wall-plug efficiency for DUV LEDs. Another advantage of our design is that, from the point of view of epitaxial growth, having the current spreading layer in the n-type electron supplier layer allows the epi-growers more freedom in optimizing the growth conditions.0.60Ga0.40N/p-AlxGax1−N/n-Al0.60Ga0.40N layer, and the Si doping concentration of the n-Al0.60Ga0.40N region is 5 × 1018 cm−3. Then, five pairs of Al0.45Ga0.55N/Al0.56Ga0.44N multiple quantum well (MQW) active layers are designed, for which the thicknesses of quantum wells and quantum barriers are 3 nm and 12 nm, respectively. The MQWs are capped by an 18-nm-thick Mg-doped p-Al0.60Ga0.40N layer serving as the p-EBL, after which a 50-nm-thick Mg-doped p-Al0.40Ga0.60N layer and a 50-nm-thick Mg-doped p-GaN layer follow. The hole concentration for the p-EBL and the hole supplier layers are set to be 3 × 1017 cm−3. We design the device geometry with a rectangular mesa of 350 × 350 μm2. Figure xGax1−N regions. The energy barriers can adjust the horizontal current distribution in the p-type hole supplier layer. Note, to guarantee the current stream through the reversely biased n-AlGaN/p-AlGaN junction, it is very important to have the p-AGaN insertion layer fully depleted so that the NPN-AlGaN junction will be in a reach-through breakdown mode [0.60Ga0.40N layer is utilized as the electron supplier layer.The NPN-AlGaN DUV LED structures are schematically drawn in Fig. own mode . DetaileI1 > I2 > I3 > … > In [I1) and a horizontal portion (I2) from point A to point B. Therefore, the total voltage between the two points is shared by the current spreading layer, the p-GaN layer, the p-AlGaN layer, the MQWs, the NPN-AlGaN junctions, and the n-AlGaN layer. Based on the current paths of I1 and I2, Eqs. RCL−V and RCL−L are the vertical and horizontal resistances for the current spreading layer, respectively; Rn−V and Rn−L denote the vertical and horizontal resistances for the n-AlGaN layer, respectively; Rn is the summation of Rn−V and Rn−L  for the current path I1; the summation of the resistance for the p-type hole injection region and MQW region is represented by Rx; Rnpn is the interfacial resistance induced by the barrier height in each NPN-AlGaN junction; N means the total number for the NPN-AlGaN junction, and the total voltage drop between points A and B is described by UAB. It is worth mentioning that the 200-nm-thick current spreading layer is much thinner than the 4-μm-thick n-AlGaN electron supplier layer for all studied devices. Therefore, a CL of which the electrical resistance is much bigger than that for n-AlGaN layer is obtained, i.e., RCL−L − Rn−L ≫ 0. It is obvious that the ratio of I1/I2 can be reduced by making N × Rnpn value increase. Hence, the current spreading effect in the p-type hole supplier layer can be improved by using the NPN-AlGaN junction in the n-type electron supplier layer for DUV LED structures. On one hand, the N × Rnpn value can be enhanced through increasing N. On the other, the value of Rnpn is affected by the AlN component, the thickness, and the Mg doping concentration for the p-AlGaN insertion layer. Therefore, a detailed analysis will be conducted in the subsequent discussions.To better understand the physical mechanism for the improved current spreading effect that is enabled by the NPN-AlGaN junction, an equivalent circuit for the DUV LED with a lateral current-injection scheme is shown in Fig. > … > In . The inc−30 cm6/s [Crosslight APSYS simulator is used to investigate the device physics, and the models that we use are reliable according to our previous publications on blue, UVA, and DUV nitride-based LEDs –24. In o30 cm6/s , 10 ns [30 cm6/s , and ~ 830 cm6/s  for DUV 30 cm6/s .0.60Ga0.40N insertion layer, for which the Mg doping concentration is 1 × 1018 cm−3. Figure 2 for LED B. Two energy barriers in the conduction band are formed in the NPN-AlGaN junctions, and the formation of the energy barrier is well ascribed to the depletion effect of the inserted p-Al0.60Ga0.40N layer. The generated barriers in LED B induce the interfacial resistance of Rnpn in the NPN-AlGaN junction region, which helps decrease I1/I2 as mentioned in Eq. I2. We then calculate and show the horizontal hole concentration in the last quantum well (LQW) for LEDs A and B when the current density is 170 A/cm2, as presented in Fig. LED A  and LED B  have been firstly investigated to probe the influence of the NPN-AlGaN structure in homogenizing the current for the p-type hole supplier layer. Each NPN-AlGaN junction has a 20-nm-thick p-AlBesides showing the lateral hole concentration, we also demonstrate the hole concentration levels in the MQWs for LEDs A and B in Fig. 2 according to Fig. 2 as shown in Fig. The impact of the NPN-AlGaN junction is also justified by the calculated optical and electrical performances for LEDs A and B as shown in Fig. i  with different NPN-AlxGax1−N junctions, for which the AlN compositions for p-AlxGax1−N insertion layers are 0.60, 0.63, 0.66, 0.69, and 0.72, respectively. The doping concentration and thickness for the p-AlxGax1−N layer are 1.8 × 1018 cm−3 and 20 nm, respectively. Two NPN-AlGaN junctions, i.e., NPNPN-AlGaN junction are used for all studied devices. We then calculate the conduction band barrier height for each NPN-AlxGax1−N junction for LEDs Ci  as shown in Table xGax1−N insertion layer increases. High conduction barrier height can make the value of Rnpn increase, and a decreased ratio of I1/I2 is simultaneously triggered as mentioned in Eq. 2 are calculated and exhibited in Fig. 0.60Ga0.40N structure is adopted, and it is obvious that the current spreading effect obtains further improvement once the AlN component of p-AlGaN insertion layer increases up to 0.63 for our structures.In this section, the impact of AlN composition for the NPN-AlGaN junction on the optical and electrical properties for DUV LEDs is studied. In order to clearly illustrate this mechanism, we use five DUV LEDs, i.e., LEDs Ci  in Fig. 2, respectively. The hole concentration levels and radiative recombination profiles are collected at the location of 120 μm away from the right edge of the mesa. We spatially shift the hole concentration levels and radiative recombination profiles in Fig. We demonstrate the simulated hole concentration levels and radiative recombination profiles in the MQWs for LEDs A and Ci  are presented in Fig. 2. It is noteworthy that the forward voltage decreases for LEDs C2, C3, and C4 when compared with LED A. Although the NPN-AlGaN junction increases the vertical resistance for DUV LEDs, the more uniform carrier concentration along the horizontal direction improves the horizontal conductivity, thus leading to a reduced forward voltage. It indicates that the enhanced current spreading effect can help to reduce the forward operating voltage for DUV LEDs as long as the current spreading layer is properly designed [The optical power density and EQE as a function of the injection current density are further calculated and exhibited for the studied LEDs in Fig. designed . Howeverdesigned , e.g., Li  can be further improved when the AlN composition of the p-AlGaN layer increases for the NPN-AlGaN junction. However, LED C2 shows the highest WPE owing to the lowest forward operating voltage despite the relatively low optical power density among LEDs Ci . In addition, we show the WPE and EQE at the injection current density of 170 A/cm2 for all investigated devices in the inset figure. It is well known that the current crowding effect is serious at a high injection current density. The NPN-AlGaN junction for LED C5 plays best in homogenizing the current. However, the WPE is not satisfactory once the forward operating voltage significantly increases. Therefore, one shall fully optimize the value of AlN component of the p-AlGaN insertion layer for the NPN-AlGaN junction before one can get the enhancement for both EQE and WPE.The WPE as a function of the injection current density for all studied devices is exhibited in Fig. Rnpn can be increased once the depletion region for NPN-AlGaN junction becomes wide, and the value of I1/I2 will be reduced, i.e., the current spreading effect for DUV LEDs can be improved. For better elucidating the point, five DUV LEDs with different Mg doping concentrations for the p-AlGaN insertion layer in the NPN-AlGaN junction have been designed and investigated. We set the Mg doping concentrations for the p-AlGaN layer to 3 × 1017, 7.5 × 1017, 1.7 × 1018, 2 × 1018, and 3 × 1018 cm−3 for LEDs Di , respectively. The thickness and the AlN composition for the p-AlGaN insertion layer are 20 nm and 0.61, respectively. We adopt two NPN-AlGaN junctions. As shown in Table 2 in Fig. The width of the depletion region for the NPN-AlGaN junction can be managed by varying the Mg doping concentration for the p-AlGaN insertion layer, and the conduction band barrier height will also change accordingly. Thus, the value of 2, respectively, and the location where the data are collected is 120 μm away from the right mesa edge. As expected, LEDs Di  have the higher hole concentration levels and radiative recombination profiles in the MQWs when compared to LED A, while the hole concentration and radiative recombination increase with the increasing Mg doping concentrations in p-AlGaN layer for the LEDs with NPN-AlGaN junctions. We contribute the rising hole concentration in the MQWs for LEDs Di  to the enhanced current spreading effect.Then, the calculated hole concentration levels and radiative recombination profiles in the MQWs are demonstrated for all studied LEDs in Fig. i  accordingly show the promoted EQE and optical power density  are illustrated in Fig. i  increase with the increasing Mg doping concentration for the p-AlGaN insertion layer. Among them, LED D5 shows the largest turn-on voltage, and this is ascribed to the parasitic diode that is caused by the very high level of Mg doping concentration in the p-AlGaN layer. According to the inset figure of Fig. 2.Owing to the reduced current crowding effect and the rising hole concentration in the MQWs, LEDs Di  are higher than that for LED A. The WPE for LED D5 exceeds that for LED A only when the injection current density is larger than 43 A/cm2. The lower WPE for LED D5 at the current density smaller than 43 A/cm2 is owing to the additional forward voltage consumption at the NPN-AlGaN junction as mentioned previously. From the inset figure, it can be seen that the EQE shows an ascending trend with the increase of Mg doping concentration for the p-AlGaN layer. However, the WPE decreases with the further increase of the Mg doping concentration for the p-AlGaN layer. Hence, we conclude that the current spreading effect and the forward voltage are very sensitive to the Mg doping level of the p-AlGaN insertion layer.For a more comprehensive analysis, we calculate the WPE as a function of the injection current density for all studied LEDs as shown in Fig. 18 cm−3, respectively. We then set different thicknesses of 18, 20, 24, 28, and 32 nm for the p-AlGaN layer in LEDs Ti , respectively. The calculated conduction band barrier heights for each NPN-AlGaN junction are presented in Table I1/I2 and correspondingly the improved current spreading.In this section, the impact of the thickness for the p-AlGaN insertion layer in the NPN-AlGaN junction on the LED performance is investigated. First of all, two NPN-AlGaN junctions  are applied for all studied DUV LEDs, of which the AlN composition and the doping concentration for the p-AlGaN layer in the NPN-AlGaN junction are 0.61 and 1.5 × 10i  when the current density is 170 A/cm2 in Fig. i  at the injection current density of 170 A/cm2. The hole concentration levels and radiative recombination profiles are collected at the location of 120 μm away from the right-hand edge of the mesa. We can see that, when compared to that of LED A in the MQWs, LEDs Ti  show the higher hole concentration levels and thus higher radiative recombination profiles. Once the thickness of the p-AlGaN layer is increased, further enhanced hole concentration and radiative recombination in the MQWs can be obtained.We calculate and show the horizontal hole concentration in the LQW for LEDs A and Ti  in Fig. i  exhibit a significant reduction when compared to that for LED A at the injection current density larger than 102 A/cm2, which is due to the significantly improved current spreading effect after adopting the NPN-Al0.61Ga0.39N junction as mentioned previously. However, a too thick p-AlGaN layer can cause an increase in the turn-on voltage owing to the parasitic N-AlGaN/P-AlGaN diode, e.g., LED T5 has the highest forward operating voltage among all the investigated LEDs when the current density is 170 A/cm2, which is also shown in the inset figure of Fig. The observed optical power density and EQE for all studied LEDs in Fig. To this end, it is particularly important to further discuss the impact of higher forward operating voltage on DUV LED performance. Therefore, we calculate the WPE for all investigate devices and show the results in Fig. 0.61Ga0.39N layer is adopted in the NPN-AlGaN junction for the proposed DUV LEDs in this section, for which the Mg doping concentration and thickness are 1.5 × 1018 cm−3 and 20 nm, respectively. LEDs Ni  have 1, 2, 3, 4, and 5 NPN-AlGaN junctions, respectively. As presented in Table i . However, the total conduction barrier height for NPN-Al0.61Ga0.39N junctions in each investigated DUV LED surely increases when more NPN-Al0.61Ga0.39N junctions are utilized. Thus, the value of N × Rnpn can be enhanced, which helps to better spread the current horizontally, i.e., the increased value of I2 in Eq. Finally, we investigate the influence of the NPN-AlGaN junction number on the current spreading effect. The p-Ali  when the current density is 170 A/cm2 are exhibited in Fig. 0.61Ga0.39N junction, and further improvement can be obtained when more NPN-AlGaN junctions are included. Ascribed to the enhanced hole concentration in the MQWs, the optical power density and EQE for the DUV LEDs with NPN-AlGaN junction also shows a significant improvement. The current-voltage characteristics for all studied devices are shown in Fig. i  are lower than that for LED A, and this indicates that the current spreading effect can help to reduce the forward voltage once the Mg doping concentration, thickness, and AlN composition for the p-AlGaN layer are appropriately applied to the NPN-AlGaN junction. The turn-on voltage for all LEDs with NPN-AlGaN junction is almost the same as that for LED A, which illustrates the negligible impact of the reversely biased N-AlGaN/P-AlGaN parasitic junction if the Mg doping concentration in the p-AlGaN layer is properly set, i.e., the p-AlGaN layer has to be completely depleted before the device is biased.Then, the hole concentration levels and radiative recombination profiles in the MQWs for LEDs Ni  in Fig. 0.61Ga0.39N junction have been promoted owing to the reduced forward operating voltage. In the inset figure, we show the EQE and WPE for LEDs A and Ni  when the current density is 170 A/cm2. Although the EQE and WPE for LEDs Ni  increase with the increasing of the NPN-AlGaN junction number, clearly, we can see that the magnitude of the increase is gradually decreasing, which indicates that the NPN-AlGaN junction number also shall be set to a proper number, and we firmly believe that the device will consume more electrical power if too many NPN-AlGaN junctions are adopted in DUV LEDs.Last but not the least, the WPEs have also been demonstrated for LEDs NTo conclude, we have suggested embedding the NPN-AlGaN junction in the n-type electron supplier layer for DUV LEDs. After comprehensive and systematic discussions, we find that the NPN-AlGaN junction can reduce the current crowding effect in the p-type hole supplier layer and improve the hole injection for DUV LEDs. The NPN-AlGaN junction can tune the conductivity for the n-type electron supplier layer so that the current path in the p-type hole supplier layer can be manipulated. For further explorations, we have investigated the impact of different parameters for NPN-AlGaN junctions on the current spreading effect, the EQE, and the WPE. We find that the current can be further homogenized if the AlN composition, the Mg doping concentration, the thickness of the p-AlGaN insertion layer, and the NPN-AlGaN junction number are increased properly. Although the EQE can be promoted by using the proposed NPN-AlGaN junctions, the WPE is not always monotonically improving, which arises from the additional voltage drop at the barriers within the NPN-AlGaN junctions. Hence, more attention shall be made when designing NPN-AlGaN current spreading layers for DUV LEDs. However, we firmly believe that our results have provided an alternative design strategy to reduce the current crowding effect for DUV LEDs. Meanwhile, we also have introduced additional device physics and hence are very useful for the community.